Hepatoprotective Activity of Cucumis trigonus Roxb. Fruitagainst CCl4 Inducesd Hepatic Damage in Rats

In India, a number of medicinal plants and their formulations are used to cure hepatic disorders in traditional systems of medicine. No systemic study has been done on protective effect of Cucumis trigonus Roxb. (Cucurbitaceae) to treat hepatic diseases. Protective action of C. trigonus fruit extracts was evaluated in this study in animal model of hepatotoxicity, which was induced by carbon tetrachloride. Forty two healthy female albino Wistar rats weighing between 180 and 200 g were divided in to seven groups of 6. Group 1 was normal control group; Group 2, the hepatotoxic group was given CCl4; Group 3 was administered standard drug (Liv-52); Groups 4-7 received pet. ether, chloroform, alcohol and aqueous fruit extract (300 mg/kg) with CCl4. The parameters studied were alanine transaminase, aspartate transaminase, alkaline phosphatase and serum bilirubin activities. The hepatoprotective activity was also supported by histopathological studies of liver tissue. Results of the biochemical studies of blood samples of CCl4 treated animals showed significant increase in the levels of serum enzyme activities, reflecting the liver injury caused by CCl4. Whereas blood samples from the animals treated with chloroform and aqueous fruit extracts showed significant and alcohol extract showed highly significant decrease in the levels of serum markers, indicating the protection of hepatic cells. The results revealed that alcoholic fruit extract of Cucumis trigonus could afford highly significant protection against CCl4 induced hepatocellular injury.


Introduction
is an organ of paramount importance as it plays an essential role in maintaining the biological equilibrium of vertebrates (1). Additionally, it is the key organ of metabolism and excretion is continuously and variedly exposed to xenobiotics because of its strategic placement in the body. The toxins absorbed from the intestinal tract of liver ailments. Thus liver diseases remain one of the serious health problems (2). Conventional or synthetic drugs used in the treatment of liver diseases are sometimes inadequate and can have serious adverse effects (3)(4)(5).
Therefore, many folk remedies from plant origin are evaluated for its possible antioxidant and hepatoprotective effects against different chemical-induced liver damage in experimental animals. CCl 4 -induced hepatotoxicity model is frequently used for the investigation of hepatoprotective effects of drugs and plant extracts. The changes associated with CCl 4induced liver damage are similar to that of acute viral hepatitis (6).
Cucumis trigonus known as ' Bitter gourd" is a plant belonging to the Cucurbitaceae family and is indigenous to india, ceylon, malaya, north australia, afghanistan and persia (7). In Indian traditional system of medicine the fruit pulp of the plant is used as expectorant, liver tonic, stomachic and purgative. The fruit pulp is useful in leprosy, jaundice, diabetes, bronchitis and amentia (8). No systematic study has been done Cucumis trigonus to treat hepatic diseases. Therefore, the protective action of Cucumis trigonus fruit extracts was evaluated in an animal model of hepatotoxicity induced by carbon tetrachloride.

Plant extract
The fruits of Cucumis trigonus Roxb. were collected in August 2008 from Beed, Maharashtra State, India. The plant was authenticated by medical research centre, belgaum, karnataka, india and the voucher specimen has been deposited in the herbarium of the Department of College of Pharmacy, Belgaum, Karnataka, India. The collected fruits were shade dried at room temperature. The two hundred grams of dried powdered fruits of Cucumis trigonus were extracted by continuous hot extraction process using soxhlet apparatus with petroleum ether, macerated with chloroform-water IP (As per ( ( Indian Pharmacopeia and concentrated under reduced pressure and low temperature (40 o C) on a rotary evaporator.

Phytochemical analysis
The extracts of the plant material were screened for various classes of natural products using standard qualitative methods as described by Harborne (9).

Experimental animals
Female albino Wistar rats weighing between 180 and 200 g were obtained from animal Government Veterinary College, Hebbal, Bangalore, India. Animals were maintained on a standard laboratory diet. Food and water were given ad libitum. They were housed in standard stainless-steel cages at a 12 h cycle of light and humidity maintained at 50%. All the chemicals used were of the analytical grade from standard companies.

Treatment of animals
Rats were randomly divided into 7 groups with 6 animals in each group. Group 1 served as negative control and was administered a single daily dose of distilled water by oral gavage for administration of CCl 4 solution in olive oil) on 1 st , 4th and 7 th day to the animal of remaining group. Group 2 received only CCl 4 , group 3 received CCl 4 and standard drug control groups (4, 5, 6 and 7) were given the plant extracts orally in doses of 300 mg/kg/0.2 after the administration of carbon tetrachloride, for 7 days (10, 11). Twenty four h after CCl 4 injection animals were anaesthetized by light ether anaesthesia and blood was collected from the vena cava, and the serum was separated for subsequent use for different enzyme measurements. The rats were then decapitated and the livers were carefully dissected and cleaned of extraneous tissues. Part of the liver tissue was immediately transferred to 10% formalin for histopathological assessments.

Assessment of liver damage
bilirubin according to the method of Reitman, Kind and Mally by using commercially available test kits (12)(13)(14).

Statistical analysis
Data were analyzed by one-way analysis of variance (ANOVA), followed by the Tuky's test for individual comparisons using SPSS software and p

Results
The yield of dried extract was pet. ether (40-1.8 (%) and aqueous 11.7 (%.).The alcohol and chloroform extracts were found to be positive for the presence of steroids, triterpenoids, saponins and glycosides. Aqueous extract was found positive for the presence of carbohydrates, steroid and triterpenoids. However pet. ether extract was found positive for the presence of fats and oils.
Administration of CCl 4 to rats caused a Treatment of rats with 300 mg/kg dose of the Cucumis trigonus alcoholic, chloroform and aqueous extracts (p.o.) markedly prevented CCl 4 bilirubin. However, 300 mg/kg of the petroleum ether extract did not prevent elevation of the enzymes. Serum bilirubin (total) levels were also 4 treatment but total bilirubin was remarkably reduced by treatment with 300 mg/kg of the alcoholic, chloroform and 4 -induced activities (Table 1).
Histopathological examinations of the liver sections of the rats treated with CCl 4 showed centrilobular necrosis, fatty changes, congestion central veins. Centrilobular necrosis, which is a more severe form of injury, was markedly prevented by treatment 300 mg/kg doses of alcohol, chloroform and aqueous extract but not 300 mg/kg dose of the petroleum ether extract (Table 2).

Discussion
In Indian system of medicine certain herbs are claimed to provide relief against liver disorders. The claimed therapeutic reputation present study one such drug Cucumis trigonus was taken for the study. The chloroform, alcohol and aqueous extract of Cucumis trigonus possess extract against CCl 4 damage. The petroleum ether extract did not protect rat liver against CCl 4 damage.
Our investigation on the extracts showed the presence of steroids, triterpenoids and cardiac glycosides in the alcoholic extract. According to these results, it maybe hypothesized that steroids and triterpenoids, which are present in the alcoholic extract, could be considered responsible for the hepatoprotective activity. CCl 4 metabolism begins with the trichloromethyl free radical (CCl 3 ) by the action of the mixed function of the cytochrome P 450 oxygenase system. This free radical, which is initially formed as relatively unreactive, reacts very rapidly with oxygen to yield a highly reactive trichloromethyl peroxy radical (CCl3OO ). Both radicals are capable of binding to proteins or lipids, or abstracting a hydrogen atom from an unsaturated lipid, thus, initiating may cause peroxidative tissue damage in cytochrome P 450 -dependent oxygenase activity could cause a reduction in the level of toxic reactive metabolites and a decrease in tissue injury. On the other hand, an elevation of plasma be regarded as a sign of damage to the liver cell membrane.
carbon tetrachloride-mediated liver injury exert their protective action by toxin mediated lipid peroxidation either via a decreased production of CCl 4 derived free radicals or through antioxidant activity of the protective agent themselves (20).

Conclusions
In conclusion, the results indicated that under the present experimental conditions, alcoholic extract of Cucumis trigonus fruit showed hepatoprotective effects against CCl 4 induced liver damage in rats.

Acknowledgement
We are thankful to Dr. F.V. Manvi, Principal, Karnataka for providing all the necessary facilities to conduct this work. Harsha Hedge, Centre, Belgaum, Karnataka, for authentication of the plant material.